Ribonucleic acid (RNA) is a nucleic acid molecule that, unlike double-stranded DNA that contains deoxyribose, is mostly single-stranded and contains ribose. RNA is inherently less stable than DNA because it is more prone to hydrolysis. This makes it very difficult to deliver RNA to a cell or an organism as it is easily broken down, rendering it biologically inactive.
Ribonucleases are enzymes that catalyze the degradation of RNA. Ribonucleases are extremely common in all cells, resulting in very short lifespans for any RNA that is not in a protected environment. Natural mechanisms have developed to protect RNA from ribonucleases including 5′ end capping, 3′ end polyadenylation, folding within an RNA protein complex, and ribonuclease inhibitor (RI). Nevertheless, the delivery of RNA to cells or organisms is greatly complicated by the presence of ubiquitous and hardy ribonucleases that degrade RNA, making it very difficult to deliver RNA to cells. Currently, RNA is mostly delivered to cells via DNA encoding said RNA, e.g. in form of vectors, so that the RNA is synthesized in situ within the cell. However, this approach requires a range of control, selection and detection molecules that need to be introduced to the cell as well. Such additional molecules complicate the production and are not always desirable due to cell toxicity concerns.
The problems connected to RNA delivery have escalated with ‘RNA interference’ (RNAi)—a genetic expression control mechanism first discovered by Fire, Mello and co-workers in the late 1990s. RNAi refers to the specific down-regulation of proteins in target cells or organs. To use such RNAi in gene therapy there is a need to deliver the RNAi to cells.
Similarly, gene expression of a multitude of undesired proteins in a cell can be altered through post-transcriptional gene silencing achieved by the introduction of small interfering RNA (siRNA) molecules into the cytoplasm of the cell.
siRNA, a double stranded 21-23 nucleotides RNA duplex having complementarity to a target mRNA, is separated the single strands, the so-called passenger strand and guide strand. While the passenger strand is degraded, the guide strand is incorporated into the RNA-induced silencing complex (RISC), binds it complementary mRNA and prevents translation thereof by means of RNAse (argonaute) activation. Due to its simplicity and the low-dose effect, RNAi can be regarded as a promising tool for an elegant, curative treatment of a wide range of diseases.
Various approaches have been reported for delivering siRNA into the cytoplasm, such as polymeric nanoparticles (NPs), liposomes and surface modifications by folate, cholesterol, biotin or fluorescent molecules (Guo et al., Adv. Drug Del. Rev. 2010, 62, 650; Kapoor et al., Int J Pharm, 2012, 427, 35; and Tan et al., Small 2011, 7, 841.)
To improve gene silencing efficiency, viral vectors have been utilized for siRNA delivery as well. Nevertheless, overcoming viral vector oncogenicity and immunogenicity remains a significant barrier for viral-based siRNA delivery (Whitehead et al., Nat. Rev. Drug Discov. 2009, 8, 129.). The poor cellular uptake of naked siRNA, its rapid degradation by RNAses and the difficulty of targeting of siRNA to systemic disease sites are currently limiting the widespread use of siRNA therapeutics (Guo et al., 2010). To overcome this, lipid or polymer-based siRNA delivery systems have been successfully used for local siRNA delivery, particularly to ocular, intradermal, liver, neural, pulmonary targets. In addition to effective cellular uptake, non-toxicity/non-immunogenicity of the carriers and effective intracellular delivery of siRNA are essential for RNAi to function as therapeutics. Therefore, current research focuses on non-viral vectors, such as polymer-based nanoparticles to overcome these challenges. However, most non-viral carriers lack acceptable efficacy and possess a high level of cytotoxicity (Tan et al., 2011).
Consequently, efficient and safe delivery systems for siRNA therapeutics remain a challenge.
The layer-by-layer (LbL) self assembly of polycations and polyanions on colloids was first described by Donath et al. (Angew. Chem. Int. Ed. Engl. 1998, 37, 2201.). The gentle assembly based on electrostatic interactions between positively and negatively charged polymers is a simple and versatile method with high applicability. Former studies focused on micro/nanoparticles such as polystyrene latex, silica and melamine formaldehyde and more biocompatible templates, such as calcium carbonate, poly(D,L-lactide-co-glycolide) (PLGA) flat templates, as well as biological cells.
Secreted protein, acidic and rich in cysteine (SPARC; also called osteonectin) is a calcium-binding extracellular matrix glycoprotein that modulates the interaction between the cell and the extracellular matrix and cell migration. There is a strong association between elevated expression of SPARC and tissue scarring and fibrosis. Increased expression of SPARC has been observed in fibrotic disorders and targeting of SPARC expression to modulate fibrosis has been evaluated as a potential therapeutic approach.
Fibrosis, which is the secretion and deposition of the cell extracellular matrix (ECM), is a frequent result of various diseases such as hypertension, diabetes, liver cirrhosis and inflammatory processes. In fibrosis, there is elevated SPARC expression indicating the involvement of the SPARC protein in modulating ECM interactions. SPARC expression and up-regulation has been reported in multiple types of fibrosis, both in human tissues and animal models. Researchers have shown that inhibition of SPARC expression decreases fibrosis involving dermal, hepatic, renal, pulmonary, intestinal fibrosis and glaucoma. Seet et al (PLoS One, 2010, 5, 9415), reported that the reduction of SPARC improved surgical success in a surgical mouse model of ocular scarring. Hence, the targeting of SPARC expression has been identified as a potential therapeutic strategy for wound modulation and reducing scarring since SPARC down-regulation also resulted in delayed cell migration, reduced collagen contractility and lower expressions of profibrotic and pro-inflammatory genes (Seet et al., J. Cell. Mol. Med. 2012, 16, 1245).
Elevated secreted protein, acidic and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis, while inhibition of SPARC can reduce scarring. siRNA targeted at the SPARC gene is therefore a promising way to inhibit SPARC and reduce scarring. The challenge is to deliver bioactive SPARC siRNA to an injury site.